Heptanal, 2-(phenylmethylene)-, (2E)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The protocol was approved by Huntingdon Life Sciences Management and by the Study Director on 15 February 2010 and by the Sponsor on 18 February 2010. Experimental start date: 22 February 2010. Experimental completion date: 12 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD Guidelines for Testing of Chemicals No. 476: "Genetic toxicology: In vitro mammalian cell gene mutation tests", 1997 under GLP condition.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(E)-2-benzylideneoctanal

Cas Number:

165184-98-5

Molecular formula:

C15H20O

IUPAC Name:

(E)-2-benzylideneoctanal

Details on test material:

- Name of test material (as cited in study report): Hexyl Cinnamic Aldehyde
- Molecular weight (if other than submission substance): 216.33
- Physical state: clear pale yellow liquid
- Analytical purity: 97.63% Cis & Trans isomers
- Batch No.: 391130110
- Expiration date of the batch: January 2011
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.6, 3.2, 6.3, 12.7, 25.4, 50.7, 101.4, 202.8*, 405.7* and 811.3* μg/mL (*precipitates seen by eye)

Mutation tests:
-S9 mix Test 1 (3 hours) 5, 10, 15, 20, 25, 30, 32.5, 35, 37.5, 40 and 45 μg/mL
+S9 mix Test 1 (3 hours) 5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL
-S9 mix Test 2 (24 hours) 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: in the presence of S9 mix

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: in the absence of S9 mix

Details on test system and experimental conditions:

Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3 hour exposures, cultures contained a total of 6 x 10^6 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24 hour exposures, cultures contained a total of 1.5 x 10^6 cells in a total volume of 5 mL.
One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition. The test substance was formulated and serially diluted in the solvent. Aliquots of 50 μL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static
humidified incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. At the end of the 24 hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentrations tested in the 3 hour exposure in the absence and presence of S9 mix were 45 and 30 μg/mL respectively, and in the 24 hour exposure in the absence of S9 mix was 50 μg/mL. The formulations being added at 1% final volume in medium.

The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls Aliquots of 100 μL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37ºC. At least four serial dilutions of the test substance were tested.
Following the 3 hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF).
The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the first main test were 30 and 27.5 μg/mL in the absence and presence of S9 mix respectively.

A second test was carried out, with a 24 hour exposure in the absence of S9 mix. Duplicate 10 mL cultures containing 3 x 10^6 cells were treated for 24 hours with 100 μL of test substance, solvent or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to
2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p, the intention being to retain at least 1 x 10^7 cells. Following this, the procedure was the same as in the 3 hour treatment. The maximum concentration assessed for mutant frequency in the second main test was 45 μg/mL.

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p < 0.001. Statistics are only reported if the Global Evaluation Factor is exceeded, and this was accompanied by a significant positive linear trend.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitate (observed by eye at the end of treatment) was observed at concentrations of 202.8 μg/mL and greater in the absence and presence of S9 mix, following a 3 hour exposure. Exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 105 to 0% and from 97 to 0% respectively.
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration tested. Exposure to concentrations from 1.6 to 811.3 μg/mL resulted in RSG values from 107 to 0%. Concentrations used in the main test were based upon these data.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 94 to 22% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. Although this assay did not reach the required 10-20% RTG it is considered valid in accordance with Moore et. al (2002) which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 30 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 27.5 μg/mL were assessed for determination of mutation frequency. RTG values from 93 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
The results obtained in response to the exposure of cultures to Hexyl Cinnamic Aldehyde in the presence of S9 mix did not demonstrate mutagenic potential. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical/acceptable solvent control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

Cultures were exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 50 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Hexyl Cinnamic Aldehyde at concentrations from 5 to 45 μg/mL were assessed for determination of mutation frequency. RTG values from 92 to 18% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test
concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Main Mutation Test 1 -3 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control ^b	A	5.22	13.48	42 (192)	165 (192)	100	79
	B	5.63	14.54	36 (192)	160 (192)
	C	5.24	15.12	43 (192)	166 (192)
	D	5.46	16.15	38 (192)	166 (192)
Hexyl Cinnamic Aldehyde 5	A	5.09	15.66	42 (192)	170 (192)	94	73
	B	5.50	13.48	41 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	4.10	13.64	45 (192)	158 (192)	65	98
	B	4.18	14.25	48 (192)	165 (192)
Hexyl Cinnamic Aldehyde 20	A	2.95	10.99	35 (192)	151 (192)	45	101
	B	2.96	10.67	30 (192)	156 (192)
Hexyl Cinnamic Aldehyde 25	A	3.01	10.58	33 (192)	149 (192)	37	108
	B	2.69	8.55	37 (192)	156 (192)
Hexyl Cinnamic Aldehyde 30	A	2.69	8.33	39 (192)	147 (192)	22	156
	B	2.27	5.32	41 (192)	136 (192)
MMS 10	A	3.64	11.41	53 (192)	45 (192)	51	937
	B	4.45	13.05	54 (192)	41 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 2. Main Mutation Test 1 - 3 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count ^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control ^b	A	165 (192)	27	13	14	52	53
	B	160 (192)	34	16	18	53
	C	166 (192)	30	15	15	50
	D	166 (192)	26	11	15	58
MMS 10	A	45 (192)	209	31	178	85	83
	B	41 (192)	215	41	174	81

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 3. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix

	Replicate	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	4.30	17.21	46 (192)	166 (192)	100	72
	B	5.30	13.18	36 (192)	169 (192)
	C	3.90	13.06	33 (192)	168 (192)
	D	4.82	13.62	41 (192)	163 (192)
Hexyl Cinnamic Aldehyde 5	A	4.73	12.81	43 (192)	163 (192)	93	75
	B	5.00	11.91	34 (192)	167 (192)
Hexyl Cinnamic Aldehyde 10	A	4.18	12.65	37 (192)	168 (192)	75	64
	B	3.67	12.49	43 (192)	171 (192)
Hexyl Cinnamic Aldehyde 12.5	A	3.00	11.87	43 (192)	166 (192)	59	77
	B	3.27	11.70	32 (192)	162 (192)
Hexyl Cinnamic Aldehyde 17.5	A	1.76 ^c	8.23	52 (192)	155 (192)	21	126
	B	1.75 ^c	8.43	55 (192)	159 (192)
Hexyl Cinnamic Aldehyde 25	A	1.49 ^c	7.24	48 (192)	162 (192)	22	92
	B	1.30 ^c	7.26	35 (192)	160 (192)
Hexyl Cinnamic Aldehyde 27.5	A	1.03 ^c	5.52	38 (192)	156 (192)	18	112
	B	1.02 ^c	4.55	23 (192)	141 (192)
BaP 1	A	4.42	12.10	30 (192)	60 (192)	86	558
	B	4.26	11.78	39 (192)	56 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)


c. Cell concentration not adjusted due to insufficient growth

BaP - Benzo[a]pyrene

Table 4. Main Mutation Test 1 - 3 Hour Treatment in the Presence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	166  (192)	26	8	18	69	61
	B	169  (192)	23	10	13	57
	C	168  (192)	24	10	14	58
	D	163  (192)	34	13	21	62
BaP 1	A	60 (192)	166	33	133	80	81
	B	56 (192)	159	29	130	82

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

BaP - Benzo[a]pyrene

Table 5. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix

Treatment/Concentration (μg/mL)	Replicate	Cell Concentration (x10^5/mL) 04 h	Cell Concentration (x10^5/mL) 24 h	Cell Concentration (x10^5/mL) 48 h	Viability Plate Count^a Day 2	Mutant Plate Count^a Day 2	Mean RTG (%)	Mean MF (x10^4)
Vehicle Control^b	A	16.60	6.85	10.91	44 (192)	170 (192)	100	59
	B	15.03	6.79	11.79	45 (192)	174 (192)
	C	14.54	6.72	11.02	40 (192)	169 (192)
	D	15.69	7.46	12.06	33 (192)	172 (192)
Hexyl Cinnamic Aldehyde 5	A	14.50	6.65	10.96	40 (192)	169 (192)	92	69
	B	14.74	5.78	12.73	34 (192)	164 (192)
Hexyl Cinnamic Aldehyde 15	A	13.16	5.82	11.87	35 (192)	167 (192)	81	70
	B	13.25	6.70	10.06	33 (192)	163 (192)
Hexyl Cinnamic Aldehyde 20	A	8.91	7.53	9.34	27 (192)	168 (192)	63	52
	B	9.17	6.70	10.86	32 (192)	172 (192)
Hexyl Cinnamic Aldehyde 25	A	6.05	4.59	11.07	38 (192)	166 (192)	27	63
	B	5.89	4.50	10.59	30 (192)	169 (192)
Hexyl Cinnamic Aldehyde 30	A	5.11	3.21	10.90	36 (192)	166 (192)	18	81
	B	5.28	3.86	9.36	26 (192)	153 (192)
MMS 10	A	12.38	7.51	7.12	47 (192)	45 (192)	48	938
	B	12.54	6.94	8.50	57 (192)	38 (192)

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Table 6. Main Mutation Test 2 - 24 Hour Treatment in the Absence of S9 Mix, Colony Size Analysis

Treatment/Concentration (μg/mL)	Replicate	Mutant Plate Count^a Day 2	Total Mutant Colonies	Large Mutant Colonies	Small Mutant Colonies	% Small Mutant Colonies	Mean % Small Mutant Colonies
Vehicle Control^b	A	170 (192)	22	8	14	64	52
	B	174 (192)	18	9	9	50
	C	169 (192)	23	12	11	48
	D	172 (192)	20	11	9	45
MMS 5	A	45 (192)	219	29	190	87	86
	B	38 (192)	232	35	197	85

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = DMSO (1% v/v)

MMS - Methyl methanesulphonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

Hexyl Cinnamic Aldehyde was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Hexyl Cinnamic Aldehyde was found to be soluble at 216.33 mg/mL in Dimethyl Sulphoxide. A final concentration of 811.3 μg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the maximum concentration that does not cause a change in osmolality of greater than 50 mOsm/kg. Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Hexyl Cinnamic Aldehyde at concentrations from 1.6 to 811.3 μg/mL, relative suspension growth (RSG) was reduced from 105 to 0% and from 97 to 0% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 107 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG). Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 30 and 27.5 μg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 22 and 18% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 45 μg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 45 μg/mL, where RTG was reduced to 18%. In all tests the concurrent vehicle and positive controls were within the acceptable historical control ranges. It was concluded that Hexyl Cinnamic Aldehyde did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.